Device and method for restoration of the condition of blood

ABSTRACT

The present invention relates to a device for extracorporeal removal of harmful agents from blood or blood com-Components, comprising full length heparin immobilized on a solid substrate by covalent end point attachment. The present invention also relates to a method for extracorporeal removal of a harmful agent from mammalian blood or blood components. The present invention further relates to a process for covalent end point attachment of full length heparin to a solid substrate.

FIELD OF THE INVENTION

The present invention relates to a device for extracorporeal removal ofharmful agents from blood or blood components, the device comprisingfull length heparin immobilized on a solid substrate by covalent endpoint attachment. The present invention also relates to a method forextracorporeal removal of a harmful agent from mammalian blood or bloodcomponents. The present invention further relates to a process forcovalent end point attachment of full length heparin to a solidsubstrate.

BACKGROUND

Sepsis is most commonly induced by a systemic infection of Gram negativebacteria and today, when infections caused by antibiotic resistantstrains of bacteria constitute a major problem, alternative methods forprevention and treatment are required. Earlier studies, in vitro and invivo, have revealed that compositions containing immobilized heparinhave prophylactic properties on microbial infections. Also, aninflammatory response caused by bioincompatibility of extracorporealcircuits is a major clinical issue and can ultimately lead to sepsis.

Heparan sulfates are proteoglycans that are present on the surface ofalmost all mammalian cells. Many microorganisms utilize heparan sulfateson the surface of the mammalian cell as receptors. Furthermore,inflammatory cells and cytokines utilize heparan sulfates on the cellsurface for binding and activation. Heparin is another proteoglycan witha molecular weight of 15-25 kDa that is isolated from proteoglycans inbasophilic granules of mast cells in mammalian tissue. Due to thestructural similarity between heparin and heparan sulfates, heparinimmobilized on a solid surface binds bacteria, virus and parasites aswell as inflammatory cells and cytokines.

The development of a pro-inflammatory state is associated with adramatically increased morbidity and mortality in a number of mammaliandiseases, including septicemia, viraemia, acute or chronic renaldisease, cardiovascular disease, hypovolemic shock, anaphylacticreactions and autoimmune disease. Tissue damage and organ dysfunctionmay be caused not only by alien microorganisms, but also bypro-inflammatory mediators released in response to such an infection ordue to surface activation by conventional extracorporeal circuits(complement activation, etc.). Cytokines (such as tumor necrosis factor,interleukin-1, interleukin-6) and non-cytokines (such as nitric oxide,platelet-activating factor, complements, and eicosonoids) may inflictcollateral tissue injury and contribute to the dysfunction of multipleorgan systems as well as to organism cell death. Components frombacteria, parasites, fungi, or viruses may evoke the activation ofpro-inflammatory cytokines through a plethora of cell-types.Inflammatory cells, including macrophages, lymphocytes, andgranulocytes, are activated. Endogenous anti-inflammatory mediators arereleased in response to the infection and act to control theoverwhelming systemic inflammatory response. First, the removal ofpathogenic microorganisms is pivotal to diminish the inflammatoryresponse. Second, the fragile balance between negative and positivefeedback on the inflammatory mediators is the key factor that modulatesthe cellular damage and influences the clinical outcome, thus making thereduction of circulating pro-inflammatory stimulii and/orpro-inflammatory cytokines a key event in controlling septiccomplications.

U.S. Pat. No. 6,197,568 discloses methods for isolation, diagnosis andtreatment of microorganisms such as flaviviruses and other hemorrhagicviruses based on the interaction of said microorganisms with heparinimmobilized on agarose. Heparin-agarose as used in U.S. Pat. No.6,197,568 comprises cleaved heparin molecules immobilized on agarose.

In Artificial Organs, 26(12):1020-1025 (2002) inflammatory cytokines areadsorbed using a heparin coated extracorporeal circuit. Theextracorporeal circuit was provided with a Baxter Duraflo II heparinsurface with electrostatically bound multi point attached heparin.

There is a demand for improved methods and devices for extracorporealtreatment of blood.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide improved devices andmethods for the extracorporeal removal of harmful agents from mammalianblood or blood components.

Another object of the present invention is to provide a device forremoval of harmful agents from mammalian blood or blood components foruse in conventional extracorporeal circulation systems for e.g.hemodialysis or oxygenation.

A further object of the present invention is to provide a method forimmobilization of full length heparin onto the device without alteringthe wanted biological activities of the heparin molecules. Furthermore,the surface shall be stable under the reaction conditions used,especially with respect to leaching of heparin.

In a first aspect thereof, the present invention provides a device forextracorporeal removal of harmful agents from blood or blood components,the device comprising full length heparin immobilized on a solidsubstrate by covalent end point attachment.

The most successful technology for heparinization of surfaces currentlyin use is the Carmeda Bioactive Surface (CBAS®). In the preparation ofCBAS® surfaces, the heparin molecules are cleaved order to providereactive groups for end point attachment of the heparin fragments tosurfaces. End point attachment of the polysaccharide is necessary tomake it possible for heparin binding moieties to get access and bind tothe heparin molecules. The mean molecular weight of the heparinmolecules attached by the CBAS® procedure is 6-9 kDa.

In a device according to the present invention, a technology for heparinimmobilization is used, wherein full length heparin molecules with amean molecular weight of more than 21 KDa are end-point attached tosurfaces. Using a process of the present invention, the amount ofheparin attached to a surface can be almost doubled as compared to thepresent CBAS® state of the art. The longer chains attached by the methodof the present invention also provides a spacer function that leads to alarger amount of accessible heparin oligomers available for heparinbinding moieties to bind to.

The present inventors have found that a full length heparin coatedsurface according to the present invention binds TNF-α much moreefficiently than a conventional surface coated with heparin fragments asgenerally employed in the prior art. In the prior art, most heparincoated surfaces have been prepared by methods that involve fragmentationof the heparin molecules in order to obtain reactive groups useful incoupling the heparin fragments to solid substrates. Previous attempts tocouple full length heparin to solid surfaces have resulted in heparinsurfaces with low surface concentrations of coupled heparin, not usefulin practical applications. Other previous attempts to couple full lengthheparin to solid surfaces have resulted in multi-point attachment of theheparin molecules to the solid substrate, which greatly reduces thebinding capacity of the heparin.

Thus, in an embodiment of the invention, the immobilized heparinmolecules have a mean molecular weight of more than 10 kDa. In anotherembodiment of the invention, the immobilized heparin molecules have amean molecular weight of more than 15 kDa. In yet another embodiment ofthe invention, the immobilized heparin molecules have a mean molecularweight of more than 21 kDa. In yet another embodiment of the invention,the immobilized heparin molecules have a mean molecular weight of morethan 30 kDa. Preferably, the immobilized heparin molecules have a meanmolecular weight within the range of 15-25 kDa. The mean molecularweight may also be higher, such as in the range of 25-35 kDa.

The mean molecular weight of the immobilized heparin molecules in adevice according to the present invention is thus significantly higherthan the mean molecular weight of the heparin molecules used in thecurrent state of the art. The full length heparin molecules used inaccordance with the present invention provide improved binding capacityfor heparin binding moieties both in terms of the amount of heparinbinding molecules that can be bound per surface area unit of the solidsubstrate, and in terms of the range of molecules that can be bound bythe surface due to the increased selection of binding motifs presentedby the immobilized full length heparin molecules.

The present invention relates to a process for the preparation ofsurfaces carrying end-point attached full length heparin, which methodresults in full length heparin coated surfaces having a high surfaceconcentration of full length heparin. The full length heparin moleculesused in the various aspects of the present invention provide asignificant increase in the binding capacity for heparin bindingentities per surface area unit as compared to the heparin surfaces ofthe prior art. The heparin is preferably covalently linked to said solidsubstrate. Covalent coupling of the heparin molecules prevent leachingof heparin into blood in contact with the heparin coated surface.Leaching of heparin has been a problem in prior art techniques employingfor example electrostatic binding of heparin to surfaces.

In a more specific embodiment, said heparin is linked to said solidsubstrate by covalent end-point attachment. Covalent attachment ofheparin to a solid substrate provides better control of parameters suchas surface density and orientation of the immobilized molecules ascompared to non-covalent attachment. The present inventors have foundthat these parameters are important in order to provide optimal bindingof heparin binding harmful agents to the immobilized heparin molecules.In an embodiment, the surface concentration of the heparin on the solidsubstrate is in the range of 1-20 μg/cm². In another embodiment, thesurface concentration of the heparin on the solid substrate is in therange of 5-15 μg/cm². Covalent end-point attachment means that theheparin is covalently attached to the solid substrate via the terminalresidue of the heparin molecule.

In an embodiment of the invention, the covalent attachment of fulllength heparin molecules to a surface is achieved by the reaction of analdehyde group of the heparin molecule with a primary amino grouppresent on the surface. An inherent property of all carbohydrates isthat they have a hemiacetal in their reducing end. This acetal is inequilibrium with the aldehyde form and can form Schiff's bases withprimary amines. These Schiff's bases may then be reduced to stablesecondary amines. In an embodiment of the inventive device, said heparinis covalently attached to said solid substrate via a stable secondaryamino group.

In an embodiment, the device is a column comprising a casing containingthe heparinized solid substrate, said column having an inlet throughwhich blood may enter the column and an outlet through which blood mayexit the column and said inlet and outlet are arranged such that bloodentering through the inlet is brought into contact with said heparinizedsolid substrate before it exits the column through the outlet.

The solid substrate of the device may preferably comprise a materialhaving a large surface area. The solid substrate of the device maycomprise microparticles or hollow fibres, but other types of solidsubstrates may also be used. The total surface area of said solidsubstrate may be in the range of 0.1-20 m², preferably in the range of0.5-3 m². In certain embodiments of the invention, the material of saidsolid substrate is selected from the group consisting of glass,cellulose, cellulose acetate, chitin, chitosan, crosslinked dextran,crosslinked agarose, cross linked alginate, polyethylene, polypropylene,polysulfone, polyacrylonitrile, silicone, fluoropolymers (such aspolytetrafluoroethylene) and polyurethanes.

The solid substrate may comprise particles or beads. In an embodiment ofthe inventive device, wherein the solid substrate is particles or beads,said particles or beads may preferably comprise a material selected fromthe group consisting of polyurethanes, polyolefins, silicones,fluoropolymers (such as polytetrafluoroethylene), poly(methylmethacrylate), glass, cross linked alginates, and cross linkedpolysaccharides, such as agarose, dextran, cellulose, chitosan andstarch. Other materials commonly used in microparticles for medicalapplications may also be employed. In another embodiment of theinvention, the solid substrate comprises a cross linked polysaccharide.

In an embodiment of the inventive device, wherein the solid substratecomprises hollow fibers, said hollow fibers may preferably comprise amaterial selected from the group consisting of polysulfones, polyamides,polynitriles, polypropylenes, cross linked alginates, and cellulose.Other materials commonly used in hollow fibers for medical applicationsmay also be employed. The hollow fiber may preferably comprise apolysulfone.

The solid substrate of the device may of course also be present in othershapes or forms providing a large surface area.

The size and porosity of the solid substrate should be selected for eachapplication or treatment so as to allow a suitable blood flow ratethrough the device at an acceptable pressure drop over the device. Forcertain applications requiring a high blood flow rate and a low pressuredrop, a larger diameter particle, pore, hollow fiber or other solidsubstrate is required. In other applications that do not require a highblood flow rate and a low pressure drop, smaller diameter particles,pores, hollow fibers or other solid substrates may be used. Thus, in anembodiment of the present invention, wherein the solid substrate ispresent in the form of particles, the particle diameter may be in therange of 10 μm to 5 mm. The particle diameter may also be in the rangeof 10 μm to 1000 μm. Generally, a particle size in the range of 20-200μm is useful, but in high flow rate applications larger particles may berequired. The solid substrate may comprise one or more hollow fibers. Inan embodiment of the present invention, wherein the solid substrate ispresent in the form of hollow fibers, the inner diameter of said fibersmay be in the range of 1 μm to 1000 μm. Generally, an inner diameter inthe range of 20-200 μm is useful, but in certain applications larger orsmaller diameter fibers may be employed.

The device of the present invention should preferably be suitablydimensioned for the blood flow rate required in the application forwhich it is intended. As non limiting examples, the blood flow rate inextracorporeal circuits for renal dialysis is generally in the range of200-500 mL/min, whereas the blood flow rate in extracorporeal circuitsfor oxygenation is generally in the range of 2000-7000 mL/min. Incertain applications, such as in extracorporeal circuits for treatmentof acute sepsis, the blood flow rate may be much lower, e.g. in therange of 1-100 mL/min.

Thus, in an embodiment, the device of the present invention is suitablefor a blood flow of 200-500 mL/min. In another embodiment, the device ofthe present invention is suitable for a blood flow of 2000-7000 mL/min.In yet another embodiment, the device of the present invention issuitable for a blood flow of 1-100 mL/min.

Local blood flow patterns in blood contacting medical devices forextracorporeal circulation are known to influence clot formation viashear activation and aggregation of platelets in stagnant zones.Consequently, the device of the present invention should be designed ina fashion that does not create these problems.

In an embodiment, the inventive device is arranged in a venous-to-venousor arterial-to-venous extracorporeal bypass circuit. Such a circuit mayfurther comprise a pump, tubing and cannulae. The device may preferablybe suitable for the required blood flow for different medicalprocedures.

In another embodiment, the inventive device comprises a pump fortransporting blood through the device. In a particular embodiment, thedevice may be presented as a stand-alone unit, which may be operatedindependently of other equipment.

In an embodiment of the first aspect of the invention, the device is acolumn arranged for use with an extracorporeal circuit. The columncomprises a casing containing the heparinized solid substrate, saidcolumn having an inlet through which blood may enter the column and anoutlet through which the blood may exit the column and said inlet andoutlet are arranged such that blood entering through the inlet isbrought in contact with said heparinized solid substrate before it exitsthe column through the outlet. The heparinized solid substrate is coatedwith covalently end-point attached full length heparin at a surfaceconcentration of approximately 10 μg/cm².

A device according to the present invention may for example be useful inthe treatment or prevention of indications such as septic shock,septicaemia, disseminated intravascular coagulation, autoimmunediseases, transplant rejection. Other clinical applications involveremoval of micro organisms (e.g. malaria, hepatitis C and HIV) andheparin-binding poisons (e.g. snake venom). Use of the inventive devicein combination with conventional circuits for extracorporealcirculation, including oxygenators and dialysis machines, will decreasemorbidity and mortality associated with long term use of such circuits.

The harmful agents of the present invention may for example bepro-inflammatory mediators, such as pro-inflammatory cells orpro-inflammatory proteins. However, the device of the present inventionis not limited to the removal of pro-inflammatory cells andpro-inflammatory proteins. Any endogenous or exogenous molecule having abinding affinity for heparin may be removed using the inventive device.Also microorganisms comprising a molecule having a binding affinity forheparin may be removed using the inventive device. Microorganisms andmolecules that may be removed from blood using a device according to thepresent invention comprise for example microorganisms selected from thegroup consisting of bacteria, viruses and parasites, along with proteinsor other molecules encoded by or associated with such microorganisms.

In an embodiment, said harmful agent is a virus. In a more specificembodiment, said virus is selected from the group consisting of herpessimplex virus type 1, herpes simplex virus type 2, Influenza A virus,cytomegalovirus and human immunodeficiency virus. In another morespecific embodiment, said virus is selected from the group consisting ofherpes simplex virus type 1 or herpes simplex virus type 2.

In another embodiment, said harmful agent is a bacterium. In a morespecific embodiment, said bacterium is selected from the groupconsisting of streptococci, such as Streptococcus pneumoniae,staphylococci, such as Staphylococcus aureus, coli, such as Escherichiacoli, pseudomonas, such as Pseudomonas aureginosa, and pneumococci, suchas Pneumococcus type 2. In a preferred embodiment, said harmful agent isStaphylococcus aureus.

In yet another embodiment, said harmful agent is a parasite. In a morespecific embodiment, said parasite is selected from the group consistingof Plasmodium falciparum and Trypanosoma cruzi.

In a further embodiment, the pro-inflammatory mediator may be aninflammatory cell selected from the group consisting of inflammatorylymphocytes, inflammatory macrophages and inflammatory granulocytes.

In yet a further embodiment, the pro-inflammatory mediator may be apro-inflammatory protein, such as a pro-inflammatory cytokine. Examplesof pro-inflammatory proteins include proteins containing heparin bindingmotifs as disclosed in J. Biol. Chem., Mar. 30, 2007; 282(13):10018-27,and proteins selected from the group consisting of tumor necrosisfactor, interleukin-1, interleukin-6, protein C, interleukin-8,high-mobility group box-1 protein or macrophage migratory inhibitoryfactor. In a more specific embodiment, said pro-inflammatory cytokine isselected from the group consisting of tumor necrosis factor alpha(TNF-α), tumor necrosis factor beta (TNF-β), interleukin-1 (IL-1), andinterleukin-6 (IL-6).

In a second aspect thereof, the present invention provides a method forextracorporeal removal of a harmful agent from mammalian blood,comprising the steps:

-   -   a) providing a sample of mammalian blood,    -   b) bringing said sample into contact with full length heparin        immobilized on a solid substrate by covalent end point        attachment, under conditions allowing binding of said harmful        agent to the heparin,    -   c) separating the sample from the solid substrate, such that        said harmful agent is at least partially retained on the solid        substrate, and    -   d) recovering said sample containing a reduced amount of said        harmful agent.

In an embodiment of the second aspect of the present invention, step b)and c) of the method are performed using a device as defined by thefirst aspect of the invention. Further embodiments of a method accordingto the second aspect of the invention correspond to those specifiedabove for the device according to the first aspect of the presentinvention regarding the harmful agent, inflammatory cell, inflammatoryprotein, mammalian blood, solid substrate and heparin immobilization.

In a third aspect thereof, the present invention provides a method fortreatment of a mammalian subject suffering from a condition caused oraggravated by a harmful agent, comprising the steps:

-   -   a) extracting blood from the subject,    -   b) bringing the extracted blood into contact with full length        heparin immobilized on a solid substrate by covalent end point        attachment, under conditions allowing binding of said harmful        agent to the heparin,    -   c) reintroducing the blood, containing a reduced amount of said        harmful agent, into the bloodstream of the subject.

In an embodiment of the third aspect of the present invention, step b)of the method is performed using a device as defined by the first aspectof the invention. Further embodiments of a method according to the thirdaspect of the invention correspond to those specified above for thedevice according to the first aspect of the present invention regardingthe harmful agent, inflammatory cell, inflammatory protein, mammalianblood, solid substrate and heparin immobilization.

In an embodiment, the methods of the invention may be used for removalof pro-inflammatory cytokines from mammalian blood.

In a fourth aspect thereof, the present invention provides an apparatusfor extracorporeal circulation of blood or blood components, comprisinga conventional extracorporeal blood treatment device and a device forremoval of harmful agents as described herein.

Treatment of conditions such as sepsis or renal failure in a patientoften involve treatment of the patient's blood in an extracorporealcircuit comprising a dialysis device. The treatment method used inextracorporeal blood treatment may itself induce activation ofinflammatory cytokines resulting in an increase of these substances inthe bloodstream of the patient under treatment. Activation of cytokinesmay for example be caused by mechanical stress imparted on the bloodduring transport through tubing, connections and components of anextracorporeal blood treatment system. Another example of where this isan important issue is in oxygenators used to oxygenate blood during forexample acute pulmonary failure after trauma. In particular, thetransport of blood through narrow diameter pores or channels may causeshear stress on the blood cells present in the blood. Also, the surfacematerial of the different components with which the blood is brought incontact may affect the activation of inflammatory cytokines in theblood. An apparatus according to the present invention decreases theproblems associated with activation of pro-inflammatory cytokinesoccurring in conventional blood treatment devices.

Embodiments of a method according to the fourth aspect of the inventioncorrespond to those specified above for the method according to thefirst aspect of the present invention regarding the harmful agent,inflammatory cell, inflammatory protein and mammalian blood.

In an embodiment of the apparatus, said conventional extracorporealblood treatment device is a device for oxygenation of blood.

In another embodiment of the apparatus, said conventional extracorporealblood treatment device is a device for hemodialysis.

Other types of conventional extracorporeal blood treatment devices arealso contemplated for use in the apparatus of the present invention.

In the apparatus of the invention, the device for removal of harmfulagents should preferably be arranged downstream of the conventionalextracorporeal blood treatment device.

In a preferred embodiment of the inventive apparatus, said device forremoval of harmful agents comprises heparin immobilized on a solidsubstrate.

The solid substrate of the device for removal of harmful agents maypreferably comprise a material having a large surface area. The solidsubstrate of the device may comprise microparticles or hollow fibres,but other types of solid substrates may also be used. The total surfacearea of said solid substrate may be in the range of 0.1-20 m²,preferably in the range of 0.5-3 m².

The solid substrate may comprise particles or beads. In an embodiment ofthe apparatus, wherein the solid substrate is particles or beads, saidparticles or beads may preferably comprise a material selected from thegroup consisting of polyurethanes, polyolefins, silicones,fluoropolymers (such as polytetrafluoroethylene), poly(methylmethacrylate), glass, cross linked alginates, and cross linkedpolysaccharides, such as agarose, dextran, cellulose, chitosan andstarch. Other materials commonly used in microparticles for medicalapplications may also be employed. In another embodiment, the solidsubstrate comprises a cross-linked polysaccharide.

In an embodiment of the apparatus, wherein the solid substrate compriseshollow fibers, said hollow fibers may preferably comprise a materialselected from the group consisting of polysulfones, polyamides,polynitriles, polypropylenes, cross linked alginates, and cellulose.Other materials commonly used in hollow fibers for medical applicationsmay also be employed.

The solid substrate of the apparatus may of course also be present inother shapes or forms providing a large surface area.

In an embodiment of the apparatus according to the present invention,wherein the solid substrate is present in the form of particles, theparticle diameter may be in the range of 10 μm to 5 mm. The particlediameter may also be in the range of 10 μm to 1000 μm. Generally, aparticle size in the range of 20-200 μm is useful, but in high flow rateapplications larger particles may be required. The solid substrate maycomprise one or more hollow fibers. In an embodiment, wherein the solidsubstrate is present in the form of hollow fibers, the inner diameter ofsaid fibers may be in the range of 1 μm to 1000 μm. Generally, an innerdiameter in the range of 20-200 μm is useful, but in certainapplications larger or smaller diameter fibers may be employed.

The device for removal of harmful agents may preferably be a columncomprising a casing containing the heparinized solid substrate, saidcolumn having an inlet through which blood may enter the column and anoutlet through which blood may exit the column and said inlet and outletare arranged such that blood entering through the inlet is brought intocontact with said heparinized solid substrate before it exits the columnthrough the outlet.

The apparatus of the present invention should preferably be suitablydimensioned for the blood flow rate required in the application forwhich it is intended. Thus, in an embodiment, the apparatus of thepresent invention is suitable for a blood flow of 200-500 mL/min. Inanother embodiment, the apparatus of the present invention is suitablefor a blood flow of 2000-7000 mL/min. In yet another embodiment, theapparatus of the present invention is suitable for a blood flow of 1-100mL/min. The device for removal of harmful agents should preferably bedesigned in a fashion that does not create clot formation via shearactivation and aggregation of platelets in stagnant zones.

The surface concentration of said heparin may preferably be in the rangeof 1-20 μg/cm², preferably 5-15 μg/cm². The immobilized heparin of saiddevice may preferably be covalently attached to the solid substrate. Theimmobilized heparin of said device is covalently attached to said solidsubstrate via stable secondary amino groups. More preferably, theimmobilized heparin of said device is covalently end point attached fulllength heparin. Thus, in an embodiment of the apparatus according to thefourth aspect of the invention, the device for removal of harmful agentsmay be a device as disclosed above in reference to the first aspect ofthe invention.

In a fifth aspect thereof, the present invention provides use of anapparatus as defined above for extracorporeal treatment of blood orblood components.

In an embodiment, an apparatus as defined as defined above is used fortreatment of a patient in need of hemodialysis. In such an embodimentthe flow rate of said blood or blood component may preferably be in therange of 200-500 mL/min.

In another embodiment, an apparatus as defined above is used fortreatment of a patient in need of oxygenation. In such an embodiment,the flow rate of said blood or blood component may preferably be in therange of 2000-7000 mL/min.

In yet another embodiment, an apparatus as defined above is used fortreatment of a patient suffering from acute sepsis. In such anembodiment, the flow rate of said blood or blood component is in therange of 1-100 mL/min.

Further uses of an apparatus of the present invention will be apparentto a person skilled in art of extacorporeal treatment of blood.

In a further aspect of the invention a process for covalent end pointattachment of full length heparin to a solid substrate is provided, saidprocess comprising the steps of:

a) providing a solid substrate having primary amino functional groups,

b) mixing said solid substrate of a) with full length heparin and areducing agent in an aqueous medium,

c) allowing the heparin to bind reductively to the amino functionalgroups, and

d) recovering the solid substrate having covalently bound full lengthheparin on its surface.

In an embodiment the initial concentration of full length heparin in themixture is in the range of 20-50 g/l. The reducing agent used in theinventive process may be any suitable reducing agent as recognized by aperson skilled in the art of organic synthesis. In an embodiment thereducing agent is NaBH₃CN. The reductive binding of step c) of theinventive process may preferably be performed at a pH-value in the rangeof 3-5. The reductive binding of step c) of the inventive process maypreferably be performed at elevated temperature. In an embodiment, saidreductive binding is performed at 60° C. for 24 h. The process mayoptionally comprise the additional step of a second addition of NaBH₃CNduring the reductive binding of step c).

As used herein the terms “blood” and “blood components” refer tomammalian whole blood, blood plasma and/or blood cells, such as forexample red blood cells or platelets. It is also contemplated that theblood or blood components may be diluted or otherwise modified.

As used herein, the term “full length heparin” means heparin orderivatives thereof, which have not been cleaved in order to obtainreactive end groups for attachment to a solid surface. The molecularweight of full length heparin as present in vivo is generallydistributed in the range of about 3-40 kDa. The molecular weight ofheparin present in commercially available heparin preparations isgenerally distributed in the range of 15-25 kDa. The mean molecularweight of full length heparin is about 21 kDa.

As used herein, the term “harmful agent” may include a microorganismcausative of diseases or disorders in mammals, such as a virus, abacterium or a parasite, as well as harmful agents symptomatic ofdiseases or disorders, such as a pro-inflammatory cytokine. Examples ofharmful microorganisms include Staphylococcus-species, HIV, hepatitis C,Dengue viruses and Plasmodium species causing malaria. The harmful agentmay for example be a virus, such as herpes simplex virus type 1, herpessimplex virus type 2, Influenza A virus, cytomegalovirus or humanimmunodeficiency virus. The harmful agent may for example be a bacteriumselected from the group consisting of streptococci, such asStreptococcus pneumoniae, staphylococci, such as Staphylococcus aureus,coli, such as Escherichia coli, pseudomonas, such as Pseudomonasaureginosa, and pneumococci, such as Pneumococcus type 2. The harmfulagent may for example be a parasite such as Plasmodium falciparum orTrypanosoma cruzi. The harmful agent may for example be an inflammatorycell such as an inflammatory lymphocyte, an inflammatory macrophage oran inflammatory granulocyte. The harmful agent may also for example beinflammatory protein, such as a pro-inflammatory cytokine, for exampletumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β),interleukin-1 (IL-1), and interleukin-6 (IL-6). The above mentionedexamples of harmful agents should not be considered as limiting for thescope of the invention. As would be readily recognized by a personskilled in the art, all types of heparin binding harmful agents may beremoved using a device or apparatus or method as disclosed by thepresent invention.

As used herein, the term “pro-inflammatory cell” means a cell, which isinvolved in inflammatory response in a mammal. Examples of “inflammatorycells” include inflammatory lymphocytes, inflammatory macrophages andinflammatory granulocytes.

As used herein, the term “pro-inflammatory protein” means a protein,such as a cytokine, released for instance in connection with microbialinfection or immunization.

As used herein, the term “cytokine” means a protein, released forinstance in connection with microbial infection or immunization,selected from the group consisting of interleukins, interferons,chemokines and tumour necrosis factors.

EXAMPLES Example 1 General Method for Quantification of SurfaceImmobilized Heparin

The principle of this method is based on the chemical reaction betweenheparin and sodium nitrite in an acidic aqueous solution. TheD-glucosamine units in heparin are converted into 2,5-anhydro D-mannosewith simultaneous cleavage of the glycosidic linkage. The terminalaldehyde group in 2,5-anhydromannose reacts with3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH) toform a colored complex in the presence of iron (III) chloride-6-hydrate(FeCl₃.6H₂O). The color intensity of the complex is measured with aspectrophotometer at a wavelength of 650 nm.

Example 2 Amination of Sephadex G 10

Sodium metaperiodate (NaIO₄, 12.0 g) was dissolved in water (1.6 L) andadded to Sephadex G 10 (100 g). The mixture was kept in the dark undershaking for 17 h. After filtration and washing with water (4 L) andfinally with 0.1 M phosphate buffer (1 L), pH 7.0. The resulting productwas suspended in a solution of 200 mL Lupasol® (5% in water) in 0.1 Mphosphate buffer, pH 7.0 (1.2 L).

The gel was stabilized by addition of an aqueous solution of NaBH₃CN.Sodium cyanoborohydride (1.0 g) in 0.1 M phosphate buffer (200 mL), pH7.0, was added to the gel mixture. The mixture was kept at roomtemperature under shaking for 24 h. The gel was filtered off and washedwith water (2 L), 0.1 M phosphate buffer pH 7.0 (2 L), water (2 L), 0.1M acetate buffer pH 4.0 (2 L) and water (2 L). The gel was air dried.

Example 3 Amination of Polyethylene Beads Etching:

Polyethylene beads (PE beads) (diameter ca 300 μm, 60 g) were washed inchloroform (200 mL) during stirring for 1 hour. The beads were collectedon a glass filter, washed with 3×50 mL of chloroform and left to dry inair. Potassium permanganate (KMnO₄, 1.2 g) was dissolved in concentratedsulfuric acid (600 mL) and the pre-washed beads were added. Thesuspension was stirred for 5 min. The beads were collected on a glassfilter and carefully washed with water (5 L) and air dried.

Amination of Etched Beads:

The following solutions were prepared:

Aqueous borate buffer: Boric acid (53.0 g) and sodium chloride (3.5 g)were added to water (5.0 L). The pH value of the resulting solution wasadjusted to 9.0 by the addition of sodium hydroxide pellets.

S1: To the aqueous borate buffer solution (5.0 L), crotonaldehyde (1.7mL) and Lupasol® (5.0 mL, 5% aq) were added, resulting in the solutionS1.

S2: Sodium chloride (146.5 g) and dextran sulfate (0.5 g) were added towater (5.0 L). The pH value of the resulting solution was adjusted to3.0 by the addition of 1 M hydrochloric acid, resulting in the solutionS2.

S3: Lupasol® (25 mL, 5% aq) was added to water (2.5 L) and pH wasadjusted to 9.0 with 1 M NaOH, resulting in solution S3.

Coating Procedure:

1. The etched PE beads were added to S1 (2.5 L). The suspension wasstirred for 10 min at room temperature.

2. The beads were collected on a glass filter and washed with water (2.5L).

3. The beads were added to S2 (2.5 L). The suspension was stirred at 60°C. for 10 min.

4. The beads were collected on a glass filter and washed with water (2.5L).

5. Step 1 was repeated with fresh S1.

6. The beads were collected on a glass filter and washed with water (2.5L).

7. Step 2 was repeated with fresh S2.

8. The beads were collected on a glass filter and washed with water (2.5L).

9. The resulting beads were added to S3 (2.5 L) and the suspension wasstirred for 10 min at room temperature.

10. The beads were collected on a glass filter and washed with water(5.0 L), resulting in aminated PE beads.

Example 4

Covalent End-Point Attachment of Nitrous Acid Degraded Heparin onto anAminated Chromatographic Gel

Sephadex G 10 (10 g), aminated as described in Example 2, was suspendedin 0.1 M acetate buffer pH 4.0 (100 mL) and nitrous acid degradedheparin (1.6 g) was added. After shaking for 15 min, NaBH₃CN (100 mg)dissolved in 0.1 M acetate buffer pH 4.0 (10 mL) was added. The reactionmixture was shaken for 24 h at room temperature and additional NaBH₃CN(100 mg) dissolved in 0.1 M acetate buffer pH 4.0 (10 mL) was added, andshaking was continued for another 24 h at room temperature.

The gel was filtered off and washed in turn with water (200 mL), 0.17 Mborate buffer pH 9.0 (250 mL) and water (2 L). The gel was air dried.

Sephadex G10 beads have an average diameter of approximately 100 μm. Arough calculation reveals that 1 cm³ contains 10⁶ beads which gives asurface area of 300 cm²/cm³. Sulfur analysis of the heparinazed Sephadexgel gave a result of 0.024% sulfur. Further, if heparin was attachedonly to the surface of the beads, the heparinized Sephadex G10 hadapproximately 7 μg heparin/cm².

Example 5

Covalent End-Point Attachment of Nitrous Acid Degraded Heparin onto anAminated PE Beads

Aminated PE beads, prepared as described in Example 3, were heparinizedas described in Example 4.

By following the procedure described in Example 1, it was determinedthat the heparinized PE beads contained 2.6 mg heparin/g beads.

Example 6

Covalent End-Point Attachment of Full Length Heparin onto an AminatedChromatographic Gel

Sephadex G 10 (10 g), aminated as in Example 2, was suspended in 0.1 Macetate buffer, pH 4.0 (45 mL), and NaCl (1.46 g) and full lengthheparin (1.6 g) was added. After shaking for 0.5 h, NaBH₃CN (100 mg)dissolved in 0.1 M acetate buffer pH 4.0 (5 mL) was added. The reactionmixture was shaken for 24 h at 60° C. After 8 h, more NaBH₃CN (100 mg)was added. The gel was filtered off and washed in turn with water (200mL), 0.17 M borate buffer pH 9.0 (250 mL) and water (2 L). The gel wasair dried.

Sephadex G10 beads have an average diameter of approximately 100 μm. Arough calculation reveals that 1 cm³ contains 10⁶ beads which gives asurface area of 300 cm²/cm³. Sulfur analysis of the heparinazed Sephadexgel gave a result of 0.037% sulfur. Further, if heparin was attachedonly to the surface of the beads, the heparinized Sephadex G10 hadapproximately 11 μg heparin/cm², i.e. approximately 36% more heparin wasimmobilized when using the full length heparin than when using thedegraded heparin (c.f. Example 4).

Example 7

Covalent End-Point Attachment of Full Length Heparin onto Aminated PEBeads

Aminated PE beads, prepared as described in Example 3, were heparinizedas described in Example 6.

By following the procedure described in Example 1, it was determinedthat the heparinized PE beads contained 2.6 mg heparin/g beads.

Example 8

Covalent End-Point Attachment of Nitrous Acid Degraded Heparin onto theInner Lumen of Hollow Fibers

In this example a pediatric haemoflow dialyzer is used. The fibers aremade of polysulfone with an inner diameter of 200 microns and a wallthickness of 40 microns. The total surface area of the blood contactingmaterial is 4000 cm² and the priming volume is 28 mL.

The amination procedure is performed as described in Example 3 for PEbeads, with the exception that the etching step is omitted. Polysulfoneis hydrophilic and does not need etching. Immobilization of heparin isperformed essentially as described in Example 4, by pumping a solutioncontaining nitrous acid degraded heparin together with NaBH₃CN into thefibers.

Because measurement of the amount of heparin is a destructive procedure,a reference dialyzer that has been heparinized under identicalconditions is sacrificed and its fibers are subjected to sulfuranalysis. The results reveal a heparin content of approx. 5 μgheparin/cm², which corresponds to a content of 20 mg heparin in thedevice.

Example 9

Covalent End-Point Attachment of Full Length Heparin onto the InnerLumen of Hollow Fibers

The experiment is performed as described in Example 8, with theexception that full length heparin is used. The results reveal a heparincontent of approx. 8 μg heparin/cm², which corresponds to a content of32 mg heparin in the device.

Example 10 Adherence of Tumor Necrosis Factor Alpha (TNF-α) in Plasma toHeparinized PE Beads

Heparinized PE beads having nitrous acid degraded heparin (prepared asdescribed in Example 5) or full length heparin (prepared as described inExample 7), were used. 200 mg beads were added to columns (1 mL) with abayonet joint lid (MoBiTec, M1002).

Samples (0.5 mL) were withdrawn with a syringe from 4 mL plasma takenfrom a human patient. The plasma samples were passed through therespective columns during 30 seconds. TNF-α content in the samplesbefore and after passage through columns was measured with a Quantikine®human TNF-α/TNFSF1A high sensitivity ELISA kit (R&D Systems) with anEVOLIS instrument (BioRad).

After passage through the column with 200 mg PE beads having nitrousacid degraded heparin, the remaining TNF-α concentration in the samplewas 4.5 pg/ml. After passage through the column with 200 mg PE beadshaving full length heparin, the remaining TNF-60 concentration in thesample was 4.1 pg/ml. Thus, the decrease in TNF-α concentration inplasma that has passed over 200 mg beads that are heparinized with fulllength heparin (Mw 20 kDa) is greater than the decrease with beads thatare heparinized with nitrous acid degraded heparin (Mw 8 KDa).

Example 11 Adherence of Antithrombin (AT) to Heparinized PE Beads

Heparinized PE beads having nitrous acid degraded heparin (prepared asdescribed in Example 5) or full length heparin (prepared as described inExample 7), were used. 200 mg beads were added to columns (2.5 mL) witha bayonet joint lid (MoBiTec, S1012).

Solutions of human antithrombin III (Octapharma), 2 IU/ml in tris buffer(pH 7.4), were added to the columns. After incubation for 15 minutes,antithrombin that had bound to low affinity sites on immobilized heparinwas removed by washing with tris buffer several times. Then, antihrombinthat had bound to high affinity sites on immobilized heparin was elutedusing large volumes of 1 mg/ml heparin in tris buffer. The content ofheparin-antithrombin complexes in the resulting eluate was determined ina Sysmex CA 1500 instrument (Sysmex) using the Berichrom AntithrombinIII reagent (Sysmex). The results are shown in Table 1.

TABLE 1 Amount of antithrombin bound to heparin MBTH assay (Example 1)Antithrombin Full length heparin 2.6 mg heparin/g beads 2.48 IU/g beadsNitrous acid degraded heparin 2.6 mg heparin/g beads 1.65 IU/g beads

From these results, it is clear that full length heparin binds 1.5 timesmore antithrombin per weight unit than nitrous degraded heparin does.

1-41. (canceled)
 42. Apparatus for extracorporeal circulation of bloodor blood components, comprising a conventional extracorporeal bloodtreatment device and a device for removal of harmful agents, whereinsaid device for removal of harmful agents comprises heparin immobilizedon a solid substrate by covalent end point attachment.
 43. Apparatusaccording to claim 42, wherein said device for removal of harmful agentsis arranged downstream of the conventional extracorporeal bloodtreatment device.
 44. Apparatus according to claim 42, wherein saidheparin is covalently end point attached full length heparin. 45.Apparatus according to claim 44, wherein said full length heparin has amean molecular weight in the range of 15-25 kDa.
 46. Apparatus accordingto claim 44, wherein said full length heparin has a mean molecularweight of more than 21 kDa.
 47. Apparatus according to claim 44, whereinthe surface concentration of said full length heparin is 1-20 μg/cm².48. Apparatus according to claim 47, wherein the surface concentrationof said full length heparin is 5-15 μg/cm².
 49. Apparatus according toclaim 42, wherein said heparin is covalently attached to said solidsubstrate via stable secondary amino groups.
 50. Apparatus according toclaim 42, wherein the total surface area of said solid substrate is inthe range of 0.1-20 m².
 51. Apparatus according to claim 50, wherein thetotal surface area of said solid substrate is in the range of 0.5-3 m².52. Apparatus according to claim 50, wherein said solid substratecomprises particles or beads.
 53. Apparatus according to claim 52,wherein the particle or bead diameter is in the range of 10-1000 μm. 54.Apparatus according to claim 52, wherein the particle or bead diameteris in the range of 20-200 μm.
 55. Apparatus according to claim 52,wherein said particle or bead comprises a material selected from thegroup consisting of polyurethanes, polyolefins, silicones,fluoropolymers, poly(methyl methacrylate), glass, cross linkedalginates, and cross linked polysaccharides, such as agarose, dextran,cellulose, chitosan and starch.
 56. Apparatus according to claim 42,wherein said solid substrate comprises one or more hollow fibers. 57.Apparatus according to claim 56, wherein the inner diameter of saidhollow fiber is in the range of 10-1000 μm.
 58. Apparatus according toclaim 56, wherein the inner diameter of said hollow fiber is in therange of 20-200 μm.
 59. Apparatus according to claim 56, wherein saidhollow fiber comprises a material selected from the group consisting ofpolysulfones, polyamides, polynitriles, polypropylenes, cross linkedalginates, and cellulose.
 60. Apparatus according to claim 42, whereinthe device is a column comprising a casing containing the heparinizedsolid substrate, said column having an inlet through which blood mayenter the column and an outlet through which blood may exit the columnand said inlet and outlet are arranged such that blood entering throughthe inlet is brought into contact with said heparinized solid substratebefore it exits the column through the outlet.
 61. A method forhemodialysis which comprises contacting blood from a patient in needthereof with the apparatus according to claim
 42. 62. A method foroxygenation of blood which comprises contacting blood with an apparatusaccording to claim
 42. 63. A method for the extracorporeal treatment ofblood or blood components which comprises contacting blood with anapparatus according to claim
 42. 64. A method for oxygenation of bloodwhich comprises contacting blood from a patient in need of thereof withthe apparatus according to claim
 42. 65. A method for the treatment ofacute sepsis which comprises contacting blood from a patient suspectedof suffering from acute sepsis to an apparatus according to claim 42.66. The method according to claim 63, wherein said blood is contactedwith said apparatus at a blood flow rate of 2000-7000 mL/min.
 67. Themethod according to claim 61, wherein said blood is contacted with saidapparatus at a blood flow rate of 200-500 mL/min.
 68. The methodaccording to claim 64, wherein said blood is contacted with saidapparatus at a blood flow rate of 1-100 mL/min.